Identification of an RNA-binding region in 18S dimethyladenosine methyltransferase DIMT1 essential for proliferation of acute myeloid leukemia cells

Aberrant assembly and function of ribosomes are implicated in cancers, including acute myeloid leukemia (AML). Here, we find that 18S rRNA methyltransferase DIMT1 is essential for AML proliferation through a non-catalytic function. We demonstrate that a positively charged cleft on DIMT1 plays an important role in RNA binding. Mutations at the identified cleft (5mutA-DIMT1) significantly weakens binding of DIMT1 to rRNA in vitro and in cells. Strikingly, the RNA-binding deficient 5mutA-DIMT1 is predominantly located in the nucleoplasm, in contrast with the primarily nucleolar localization of wild-type DIMT1. Furthermore, we demonstrate that rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation in vitro, which likely promotes DIMT1's nucleolar localization and its role in rRNA processing in cells. Importantly, re-expression of wild-type but not 5mutA-DIMT1 supports AML proliferation in competition-based cell proliferation assays. These results highlight the importance of targeting the catalytically independent RNA-binding activity of DIMT1 for therapy of AML. Overall design: Ribosome profiling followed by high-throughput sequencing (ribo-seq) in MOLM13C cells with DIMT1 or Rosa (a negative control) depletion.

核糖体的异常组装与功能异常与包括急性髓系白血病（acute myeloid leukemia, AML）在内的多种癌症密切相关。本研究发现，18S核糖体RNA甲基转移酶（18S rRNA methyltransferase, DIMT1）可通过非催化功能对急性髓系白血病的细胞增殖起到必需作用。我们证实，DIMT1表面的带正电荷裂隙在RNA结合过程中发挥关键作用。针对该裂隙引入突变得到的5mutA-DIMT1，可在体外及细胞内显著削弱DIMT1与核糖体RNA的结合能力。值得注意的是，丧失RNA结合能力的5mutA-DIMT1主要定位于核质中，而野生型DIMT1则主要定位于核仁。此外，我们证实，DIMT1在体外发生液-液相分离（liquid-liquid phase separation）需要依赖其与核糖体RNA的结合能力，这一过程或可促进DIMT1的核仁定位以及其在细胞内参与核糖体RNA加工的功能。重要的是，在基于竞争法的细胞增殖实验中，重新表达野生型DIMT1可恢复急性髓系白血病细胞的增殖能力，而重新表达5mutA-DIMT1则无此效果。上述研究结果凸显了靶向DIMT1的非催化型RNA结合活性以治疗急性髓系白血病的重要价值。实验整体设计：在DIMT1或Rosa（阴性对照）敲低的MOLM13C细胞中开展核糖体谱分析结合高通量测序（ribo-seq）。